JP6483083B2 - Methods of enhancing the delivery of therapeutic compounds to the eye - Google Patents

Description

RELATED APPLICATIONS This application claims priority to and the benefits of US Provisional Patent Application No. 61 / 785,015, filed March 14, 2013, the contents of which are hereby incorporated by reference in their entirety. Be incorporated.

GOVERNMENT SUPPORT This invention was made with United States Government support under National Institutes of Health / National Eye Institute grant NIH EY 17130. The government has certain rights in the invention.

FIELD OF THE INVENTION The present invention relates generally to methods of enhancing the delivery of therapeutic compounds to the eye.

BACKGROUND OF THE INVENTION The eye is a complex optical system that detects light, converts it into a series of electrical signals, and transmits these signals to the brain, ultimately producing a representation of our world . Eye diseases and disorders can cause diminished vision, diminished light sensitivity, and blindness.

  Delivery of therapeutic compounds to specific ocular tissues affected by ocular diseases or disorders, such as the retina, is a challenge. Current methods, such as intravitreal injection or implanted drug delivery devices, still have limited delivery efficacy. Specifically, the therapeutic agent often localizes only to the adjacent area around the delivery site, and can not penetrate or diffuse across intervening ocular structures or through the target ocular tissue, thereby causing such Significantly limit the effectiveness of the treatment. Thus, there is a need for many years for methods to enhance the delivery of therapeutic compounds to the eye.

  The present invention provides a solution to the longstanding need for methods to enhance or improve the delivery of therapeutic compounds to the eye.

  The invention features plasmin or a derivative thereof and methods by administering the therapeutic agent to the eye that enhances delivery of the therapeutic agent to the subject's eye. The invention also features the use of a composition comprising plasmin or a derivative thereof for delivery to the eye of a subject to enhance delivery of a therapeutic agent.

  In one aspect, plasmin or a derivative thereof is miniplasmin or microplasmin (ocryplasmin). Plasmin or a derivative thereof included in the present invention has the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or a functional variant or fragment thereof.

  In one aspect, the therapeutic agent is selected from small molecules, nucleic acids, antibodies, or peptides. The nucleic acid is a nucleic acid expression vector (ie, a viral vector), a plasmid, or a siRNA. For example, the viral vector is an AAV viral vector encoding a transgene (ie, recombinant AAV or rAAV). Preferably, the transgene encodes a gene product that increases or restores light sensitivity, increases light detection, increases light sensitivity, increases the visual evoked potential, or restores vision to the blind. More preferably, the transgene is an opsin gene. Examples of opsin genes are channelrhodopsin (ie, channelrhodopsin-1, channelrhodopsin-2, Volvox carteri channelrhodopsin 1 or 2), melanopsin, pineal opsin, photopsin, halorhodopsin, bacteriorhodopsin , Proteorhodopsin, or any functional variant or fragment thereof, but is not limited thereto.

  Other examples of therapeutic agents include, but are not limited to, ranibizumab antibody FAB (Lucentis), VEGF Trap fusion molecule (VEGF Trap-Eye), macugen pegylated polypeptide (pegaptanib), and bevacizumab (Avastin). Any of the therapeutic agents used in the present invention may be encapsulated in nanoparticles, polymers or liposomes.

  In one aspect, plasmin or a derivative thereof and a therapeutic agent are delivered simultaneously or sequentially.

  The present invention provides methods in which a therapeutic agent is delivered to retinal cells. The retinal cells are retinal ganglion cells, retinal horizontal cells, retinal bipolar cells, axonal cells, photoreceptor cells, Mueller glial cells, or retinal pigment epithelial cells.

  In one aspect, plasmin or a derivative thereof and a therapeutic agent are administered to the vitreous of the eye.

  The invention further provides a method of increasing or restoring light sensitivity in a subject, comprising administering a plasmin or derivative thereof and a viral vector encoding opsin to the vitreous of the eye. The present invention also provides a method for improving or restoring visual acuity in a subject, comprising administering plasmin or a derivative thereof and a viral vector encoding opsin to the vitreous body of the eye.

  Also provided herein is the use of a composition comprising plasmin or a derivative thereof for treating an ocular disease or disorder in a subject.

  The subject suffers from an eye disease or disorder. Preferably, the eye disease or disorder is associated with photoreceptor degeneration.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting.

[Invention 1001]
A method of enhancing the delivery of a therapeutic agent to a subject's eye, comprising administering plasmin or a derivative thereof and the therapeutic agent to the eye.
[Invention 1002]
The method of the invention 1001, wherein the plasmin or derivative thereof is miniplasmin or microplasmin (occliplasmin).
[Invention 1003]
The method of the invention 1001, wherein the therapeutic agent is selected from nucleic acids, small molecules, antibodies, or peptides.
[Invention 1004]
The method of the invention 1003, wherein the nucleic acid is a nucleic acid expression vector, a plasmid, or a siRNA.
[Invention 1005]
The method of the invention 1004, wherein the nucleic acid expression vector is a viral vector comprising a transgene.
[Invention 1006]
The method of the invention 1005, wherein the transgene is opsin.
[Invention 1007]
The method of the invention 1006, wherein the opsin is selected from the group consisting of channelrhodopsin, halorhodopsin, melanopsin, pineal opsin, bacteriorhodopsin, and proteorhodopsin, or a functional variant thereof.
[Invention 1008]
The method of any of the present invention 1004-1007, wherein said transgene is operably linked to a cell specific promoter.
[Invention 1009]
Any of the above methods of the invention, wherein the therapeutic agent is encapsulated in nanoparticles, polymers or liposomes.
[Invention 1010]
Any of the above-mentioned inventions, wherein the therapeutic agent is selected from the group consisting of ranibizumab antibody FAB (Lucentis), VEGF Trap fusion molecule (VEGF Trap-Eye), macugen pegylated polypeptide (pegaptanib), and bevacizumab (Avastin). the method of.
[Invention 1011]
The method of the invention 1001, wherein the subject suffers from an eye disease or disorder.
[Invention 1012]
The method of the invention 1001, wherein plasmin or a derivative thereof and a therapeutic agent are delivered simultaneously or sequentially.
[Invention 1013]
The method of the invention 1001, wherein the therapeutic agent is delivered to retinal cells.
[Invention 1014]
The method of the invention 1013, wherein the retinal cells are retinal ganglion cells, retinal bipolar cells, retinal horizontal cells, axillary cells, photoreceptor cells, Mueller glial cells, or retinal pigment epithelial cells.
[Invention 1015]
The method of the invention 1001, wherein the administration is to the vitreous of the eye.
[Invention 1016]
A method of increasing light sensitivity, or improving or restoring visual acuity in a subject, comprising administering plasmin or a derivative thereof and a viral vector encoding opsin to the vitreous of the eye.
[Invention 1017]
The method of the invention 1016 wherein the opsin is selected from the group consisting of channelrhodopsin, halorhodopsin, melanopsin, pineal opsin, bacteriorhodopsin, and proteorhodopsin, or a functional variant thereof.
[Invention 1018]
The method of any of inventions 1016-1017, wherein the subject has an eye disease or disorder.
Other features and advantages of the present invention are believed to be apparent from, and encompassed by, the following detailed description and claims.

Glass of AAV2 vector (6 × 10 ¹² vg / ml), AAV2 / 2-ChR2-GFP-WPRE-hGHpA in co-injection (C, D) with control (A, B) or plasmin (0.025 IU / eye) 7 is a series of representative GFP fluorescence images of retinal vertical sections after internal injection. The vector was co-injected with plasmin into the vitreous cavity of approximately one month old adult C56BL / 6J mice. Transduction efficiency was assessed one month after virus injection by immunostaining and cell counting. FIG. 7 is a series of representative GFP fluorescence and DAPI staining images demonstrating the effect of plasmin on AAV-mediated transduction efficiency and potential neurotoxicity in retinal ganglion cells. AAV2 vector (2 × 10 ¹² vg / ml) AAV2 / 2-ChR2-GFP-WPRE-hGHpA was injected in control (A, E) or (B, F) low concentration (L: 0.005 IU) , (C, G) intermediate concentrations (M: 0.025 IU), and (D, H) high concentrations (H: 0.100 IU plasmin / eye) at the same time. Retinal ganglion cells were stained by DAPI (A to D). FIG. 2 is two graphs showing quantitative assessment of the effect of plasmin on (A) AAV-mediated transduction efficiency in retinal ganglion cells and (B) potential neurotoxicity of plasmin. Co-injection of plasmin at low (L: 0.005 IU), intermediate (M: 0.025 IU), and high (H: 0.100 IU plasmin / eye) co-injection with AAV-mediated transduction efficiency in retinal ganglion cells Significantly increased (A). Co-injection of plasmin did not show any significant neurotoxicity to retinal ganglion cells. The ganglion cell number was evaluated from multiple unit areas of 223 μm × 167 μm. ^* P <0.05; ^** p <0.005. FIG. 7 is a series of representative fluorescence images of mCherry-expressing retinal bipolar cells in retinal whole-mount. An AAV2 vector (2 × 10 ¹² vg / ml) with a Y444F capsid mutation carrying mCherry under the control of the mGluR6 promoter, in three doses (approximately 1 month old adult C56BL / 6J mice) L: 0.005 IU, M: 0.025 IU, and H: 0.100 IU / eye) co-injected with plasmin. Transduction efficiency was assessed one month after virus injection by immunostaining and cell counting. 3 is three graphs showing quantitative data for the effect of plasmin on AAV-mediated transduction efficiency in retinal bipolar cells. A) center; B) middle region; and C) periphery. The number of retinal bipolar cells expressing mCherry was estimated from multiple unit areas of 223 μm × 167 μm. ^* P <0.05; ^** p <0.01; ^*** p <0.001.

DETAILED DESCRIPTION The present invention provides methods for enhancing the delivery of a therapeutic compound or agent to the eye of a subject by administering plasmin or a derivative thereof and the agent to the eye. In some embodiments, plasmin or a derivative thereof and a therapeutic agent may be delivered to the vitreous for enhanced delivery to retina and retinal cells. Retinal cells include, for example, photoreceptor cells (eg, rods, pyramidal bodies, and photosensitive retinal ganglion cells), horizontal cells, retinal bipolar cells, axodon cells, retinal ganglion cells, Mueller glial cells, and Contains retinal pigment epithelial cells. In other embodiments, plasmin or a derivative thereof and a therapeutic agent may be delivered to, for example, the posterior segment, the anterior segment, the sclera, the choroid, the conjunctiva, the iris, the lens, or the cornea.

  The retina is a complex tissue at the back of the eye that contains specialized photoreceptor cells called rods and cones. Photoreceptors connect to networks of neurons for local processing of visual information. This information is sent to the brain for decoding into a view. The retina is associated with age-related macular degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa (RP), glaucoma, and other hereditary retinopathy, uveitis, retinal detachment, and cancer of the eye It is susceptible to a variety of diseases, including intraocular melanoma and retinoblastoma). Each of these can result in loss of vision or complete blindness.

  Delivery of therapeutic compounds to the retina is a challenge due to the complex structure of the eye. Intravitreal injection and vitreous delivery devices are frequently used to deliver therapeutic compounds to the retina, but the efficiency of delivery is impaired by the inner limiting membrane (ILM) of the retina and the cells in multiple layers.

Plasmin Plasmin is a serine protease present in the blood that degrades fibrin clots and other plasma proteins. Specifically, plasmin cleaves fibrin, fibronectin, thrombospondin, laminin, proaccelerin, and von Willebrand factor (VWF) into soluble products. Plasmin is more selective than trypsin and shows preferential cleavage on the carboxyl side of lysine and arginine residues.

Specifically, plasmin originates from an enzyme precursor called plasminogen (PLG), ie an inactive precursor protein. The amino acid sequence of plasminogen, such as Genbank Accession No. NP_000292, is known in the art and is listed below.

Plasminogen nucleic acids, such as Genbank Accession No. NM_000301, are known in the art and as listed below.

The signal peptide sequence of plasminogen is 19 amino acids in length. Thus, plasminogen sequences that do not contain a signal peptide include amino acids 20-810 of the plasminogen sequence. The signal peptide sequence is as follows.

Plasmin heavy chain A is 561 amino acids and comprises the amino acid sequence provided below.

The short form of plasmin heavy chain A is 483 amino acids. The amino acid sequence of the short form of plasmin heavy chain A is as follows:

The amino acid sequence of the activation peptide comprises the following amino acid sequence:

Plasmin light chain B is 230 amino acids. The amino acid sequence of plasmin light chain B is as follows.

  In some preferred embodiments, plasmin can be miniplasmin or microplasmin or a derivative thereof. Miniplasmin and microplasmin, upon activation of miniplasminogen and microplasminogen, include but are not limited to streptokinase, staphylokinase, tissue-type plasminogen activator, or urokinase etc. Produced by beta. Miniplasminogen and microplasminogen are derived from plasminogen, a single-chain glycoprotein that is an important component of mammalian blood. Human plasminogen consists of 791 residues consisting of an N-terminal pre-activation domain, five homologous kringle domains each of about 80 amino acids, a serine protease catalytic domain, and an interdomain connection sequence. Group multidomain protein (SEQ ID NO: 1). The plasmin or plasminogen activator is between N-terminal Arg-68 and Met-69 of human plasminogen, or Lys-77 and Lys-78, or Lys-78 and Val-79. Cleavage of the peptide bond between them resulting in a shorter proenzyme called Lys-plasminogen (e.g. a protein consisting of amino acids 69-791 or 78-791 or 79-791). Further cleavage by the enzyme elastase removes the first four kringle domains to produce the proenzyme miniplasminogen (typically amino acids 442 to 791). Further cleavage of the fifth kringle results in the proenzyme microplasminogen (typically amino acids 543-791). The plasminogen kringles contain a lysine binding site that mediates specific binding of plasminogen to a substrate such as fibrin. The proenzyme form of plasminogen is activated to the enzymatically active form by cleavage of the peptide bond between Arg-561 and Val-562, resulting in the disulfide linked duplex form of the corresponding protein It occurs. The product of activation of plasminogen protein is called plasmin. Thus, the product of Lys-plasminogen activation is referred to as Lys-plasmin, while the products of miniplasminogen and microplasminogen activation are referred to as miniplasmin and microplasmin, respectively. Miniplasmin and microplasmin, like plasmin, have catalytic activity. The advantage of miniplasmin and microplasmin over plasmin is its smaller size compared to plasmin. Thus, both microplasmin and miniplasmin are expected to have faster diffusion rates in the vitreous than plasmin.

  The plasmin of the invention comprises any one of the plasminogen related sequences described herein, eg, SEQ ID NOs: 1 and 3-7, or any one of its functional fragments or variants May be.

軽鎖のアミノ酸配列は、以下の通りである。

本発明はさらに、オクリプラスミンのバリアントおよび誘導体を包含する。 The plasmin may also be ochryplasmin (JETREA®) or a variant or derivative thereof. Ocriplasmin is a recombinant truncated form of human plasmin produced by recombinant DNA technology in the Pichia pastoris expression system. Ocriplasmin is a protein composed of 249 amino acids with a molecular weight of 27.2 kDa and has two peptide chains. The amino acid sequence of the truncated heavy chain is as follows:

The amino acid sequence of the light chain is as follows.

The invention further encompasses variants and derivatives of oculiplasmin.

  Plasmin may be isolated from blood using known isolation techniques. Alternatively, plasminogen is isolated from blood using known isolation techniques and then incubated with a protease that cleaves plasminogen into active plasmin and is suitable for use in the methods described herein. It may produce purified or isolated plasmin.

  The plasmin may be chemically synthesized using a commercially available peptide synthesizer to produce purified or isolated plasmin suitable for use in the methods described herein. Chemical synthesis of peptides and proteins can be used for incorporation of modified or unnatural amino acids, including D-amino acids and other small organic molecules. Substitution of one or more L-amino acids in a peptide or protein with the corresponding D-amino acid isoforms to increase resistance to enzymatic hydrolysis and one or more properties of biological activity, ie Can be used to enhance the duration of functional ability or action. Other modifications to plasmin can be introduced, for example, crosslinks to alter conformation to alter plasmin's ability, selectivity, or stability.

  For example, plasmin may be purchased from commercial vendors such as Sigma Aldrich, catalog number P1867. The invention also encompasses variants or derivatives of plasmin supplied by Sigma Aldrich (Catalog No. P1867).

  The plasmin may be recombinant plasmin obtained by methods well known in the art for expression and purification of recombinant proteins. DNA molecules encoding plasmin or variants or analogs thereof can be generated from known DNA sequences or by deducing nucleic acid sequences from amino acid sequences based on known codon usage. The DNA molecule encoding plasmin can be cloned into a suitable vector, such as a cloning vector or expression vector, by any of the methodologies known in the art. A cloning or expression vector is required for the expression of components required for further cloning of plasmin DNA, such as restriction enzyme sites, or for plasmin, such as host specific promoters and optionally enhancer sequences. Contains all of the components. An expression vector can be introduced into host cells for expression. Host cells can be any prokaryotic or eukaryotic cell. For example, plasmin can be expressed in bacterial cells (ie, E. coli), yeast, insect cells (ie, Sf9), or mammalian cells. Other suitable host cells are known to those skilled in the art. Host cells can be used to produce or overexpress plasmin, a variant or derivative thereof in culture. The biologically expressed plasmin, variant or derivative thereof, is then purified using known purification techniques such as affinity chromatography, and purified or suitable for use in the methods described herein. It may produce isolated plasmin.

  In some embodiments, variants, derivatives, or analogs of plasmin may be preferred. Variants, derivatives and analogues of plasmin can be identified or generated by one skilled in the art. Plasmin variants, derivatives, and analogs can be generated, for example, by using the recombinant or synthetic methods described herein. Plasmin derivatives known in the art include miniplasmin and microplasmin, which are also suitable for use in the methods disclosed herein.

  As used herein, the terms "derivative" and "variant" may be used interchangeably and refer to plasmin which is different from naturally occurring plasmin but retains its essential properties . For example, the plasmin derivative may be a biologically active fragment of plasmin, such as a truncated plasmin. The biologically active fragment contains the catalytic domain of plasmin and has serine protease catalytic activity. Alternatively, the plasmin derivative is at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, at least twenty amino acids, It may be mutated plasmin, wherein at least 30 amino acids, at least 40 amino acids, or at least 50 amino acids are mutated from wild-type plasmin. A mutated plasmin may have a sequence that has been altered by substitution, addition, or deletion that still results in a functionally equivalent molecule. In one embodiment, the plasmin derivative is about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 85%, about 80%, about 75%, about 75% with wild type plasmin. 70%, about 65%, about 60%, about 55%, or about 50% identical. In some cases, mutations may increase the capacity of plasmin (protease activity), stability, or number of targets. In another aspect, mutations may reduce the capacity, stability or number of targets of plasmin. The mutations may be conservative amino acid substitutions. Alternatively, the mutations may be non-conservative amino acid substitutions. In another aspect, plasmin derivatives may be chemically modified, for example by the addition of chemical moieties that alter activity or stability.

  The term "% identical" in the context of two or more nucleic acid or polypeptide sequences compares and aligns for maximum correspondence as measured by one of the following sequence comparison algorithms or by visual inspection: When referred to, two or more sequences or subsequences having a particular percentage of amino acid residues or nucleotides that are the same or the same. For example,% identity refers to the entire length of the coding region of the sequences being compared.

  For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test sequences and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the designated program parameters. Percent identity is determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J MoI Biol 215: 3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25 : 17, 3389-402).

  The term "analog" as used herein refers to a compound or peptide which retains the essential protease activity of plasmin. For example, the analogue may have some amino acid sequence similarity or no sequence similarity to naturally occurring plasmin. However, all plasmin analogues have a functional ability to cleave any one of the plasmin targets (ie laminin, fibrin) and protease cleavage, and / or preferential cleavage at the carboxyl side of lysine and arginine residues Hold.

  Derivatives and analogs of plasmin can be identified by those skilled in the art by screening combinatorial libraries of plasmin variants, or general therapeutic or pharmaceutical compounds.

Methods of Use The present invention provides methods for enhancing the delivery of therapeutic agents to the eye. The methods of the present invention comprise administering plasmin and a therapeutic agent to the eye. The therapeutic agent may be delivered to the eye by any method known in the art. Routes of administration include, but are not limited to, intravitreal, intravaginal, subconjunctival, subtenon, retrobulbar, posterior juxtascleral, or topical. Methods of delivery include, for example, injection through a syringe and a drug delivery device such as an implanted vitreous delivery device (ie, VITRASERT®).

  Preferably, the therapeutic agent is administered to the vitreous by intravitreal injection for delivery of the therapeutic agent to the retina. In some embodiments, the methods of the invention provide enhanced delivery to cells of the retina. Exemplary retinal cells include photoreceptor cells (eg, rods, cones, and photosensitive retinal ganglion cells), horizontal cells, bipolar cells, ataxic cells, retinal ganglion cells, Mueller glial cells, and Including but not limited to retinal pigment epithelial cells.

  In one embodiment, plasmin or a derivative thereof is administered simultaneously or sequentially with the therapeutic agent. For co-administration, plasmin or a derivative thereof can be formulated with the therapeutic agent in a single composition suitable for delivery, eg, injection, by methods known in the art. Alternatively, plasmin or a derivative thereof can be injected in separate compositions simultaneously or sequentially. In a preferred embodiment, plasmin may be administered prior to administration of the therapeutic agent.

  Such formulations may be pharmaceutically and / or physiologically acceptable vehicles, diluents, carriers or excipients such as buffered saline or other buffers, such as HEPES, to maintain physiological pH. Containing agents. For a discussion of such components and their formulations, see generally Gennaro, AE., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 2003 edition or latest edition. See also WO 00/15822. If the preparation is to be stored for a long period of time, it may be frozen, for example, in the presence of glycerol.

  The dosage of plasmin or derivative thereof to be administered can be optimized by those skilled in the art. Delivery to certain target ocular tissues may require lower doses of plasmin or higher doses of plasmin, depending on the location of the target tissue, the intervening ocular structures, and the ability of the agent to penetrate the target tissue. Preferably, the dose of plasmin administered is about 0.001 UI (enzyme unit) per eye, 0.025 UI per eye, about 0.05 UI per eye, about 0.075 UI per eye, about 0.100 UI per eye, about 0.150 UI per eye, Or about 0.200 UI per eye.

  In some embodiments, the methods for enhancing delivery disclosed herein can provide an increase in the efficacy of the therapeutic agent. An increase in the efficacy of the therapeutic can be determined by measuring the therapeutic effect of the therapeutic. Treatment is effective in providing a clinical benefit, such as alleviation of symptoms in a subject. For example, in degenerative retinal diseases such as retinitis pigmentosa, the treatment is effective if another aspect of light sensitivity or vision is improved or restored. When the treatment is applied prophylactically, "effective" means that the treatment retards or prevents an eye disease or disorder or prevents one of the clinical symptoms of an eye disease or disorder Means to do or relax. Efficacy is determined in relation to any known method for diagnosing or treating a particular eye disease or disorder.

  In some embodiments, the therapeutic agent is a nucleic acid or nucleic acid expression vector encoding a therapeutic transgene (ie, a viral vector) or a siRNA species (ie, a short hairpin or microRNA). For such therapeutic agents, the enhanced delivery of therapeutic agents provided by the methods disclosed herein may result in an increase in transduction efficiency. An increase in transduction efficiency can be determined by measuring the level of expression of the transgene introduced by the viral vector.

Therapeutic Agents Therapeutic agents delivered to the eye by the methods described herein are used in the art to treat, alleviate, reduce or prevent eye disease, eye disorders, or conditions of the eye condition. Are any of the known therapeutic agents. The therapeutic agent may be a small molecule, a nucleic acid, an antibody or a peptide.

  Examples of small molecules suitable for use in the methods described herein include, but are not limited to, tyrosine kinase inhibitors, antibiotics, anti-inflammatory agents, glucocorticoids, opioid antagonists, and other enzyme inhibitors I will not.

  Examples of nucleic acids suitable for use in the methods described herein are viral vectors encoding a therapeutic transgene (ie, channel opsin, or halorhodopsin), RNA interference molecules (ie, short hairpins, siRNA, or (Including microRNAs), but is not limited thereto. In a particularly preferred embodiment, the therapeutic agent is a viral vector encoding a transgene for gene therapy. Particularly preferred viral vectors are rAAV vectors encoding for channel opsin or halorhodopsin for expression in the retina to restore light sensitivity.

  Examples of antibodies suitable for use in the methods described herein include ranibizumab (Lucentis®), VEGF antibody (Eylea®), bevacizumab (Avastin®), infliximab, etanercept, And adalimumab, but is not limited thereto.

  Examples of proteins or peptides suitable for use in the methods described herein include microplasmin (Ocriplasmin, Jetrea®), macugen pegylated polypeptide (pegaptanib), and integrin peptides, although these It is not limited to. In some aspects, the peptide therapeutic is a collection of peptides containing two or more peptides.

  Any of the therapeutic agents described herein may optionally be encapsulated in a carrier such as nanoparticles, polymers, or liposomes. These carrier agents may serve to further enhance the delivery of the therapeutic agent to the eye. In some aspects, the carrier agent may alter the properties of the therapeutic agent, such as increasing stability (lifetime) or providing sustained release properties to the therapeutic agent. Alternatively, the carrier may protect the therapeutic agent from the proteolytic activity of plasmin when formulated in the same composition for delivery.

Gene Therapy Because many eye diseases and disorders result from aberrant gene expression in various ocular tissues, gene therapy has the potential to increase as an effective therapy. However, the efficacy of gene therapy in the eye is limited due to the task of effective delivery and transduction of therapeutic viral vectors across any ocular tissue.

  Thus, the present invention provides methods for increasing the efficiency of delivery of transgenes to the eye to treat ocular diseases or disorders, or to restore or improve vision. The transgenes of particular interest for photosensitivity or vision recovery include photoproteins such as opsin genes or rhodopsin genes. As used herein, a "transgene" is a polynucleotide encoding a polypeptide of interest, and is present in a nucleic acid expression vector suitable for gene therapy (eg, a viral vector such as AAV) Refers to a polynucleotide.

  Previous studies have shown that injection of a recombinant adeno-associated virus vector encoding a transgene such as channel opsin-2 results in poor delivery of the vector and low expression of Chop2 in retinal lining cells, especially bipolar cells It is shown. In non-human primates, AAV-mediated gene transfection has been found to be more efficient in the peripheral retina, fovea and along blood vessels, at the interface between the retina and the vitreous cavity It has been suggested that some internal limiting membrane (ILM) is a major barrier (Ivanova et al., 2010).

  The present invention provides a solution to this problem by using plasmin or a derivative thereof to lyse components of ILM such as laminin and fibronectin. Thus, the therapeutic agent will have greater accessibility to the retina, specifically to cells of the inner retina such as retinal bipolar cells, retinal ganglion cells, Muller glial cells, and retinal pigment epithelial cells. . The methods described herein provide enhanced delivery of therapeutic compounds such as therapeutic viral vectors. Enhanced delivery of the viral vector results in increased transduction efficiency of the therapeutic compound, increased expression of the therapeutic transgene (i.e., Chop2), and increased efficacy of the therapeutic compound (i.e., increased light sensitivity or visual acuity). Demonstrated by the recovery of

  Nucleic acid expression vectors suitable for use in gene therapy are known in the art. For example, the nucleic acid expression vector is a viral vector. The viral vector can be a retroviral vector, an adenoviral vector, an adeno-associated vector (AAV), or a lentiviral vector, or any engineered or recombinant viral vector known in the art. Particularly preferred viral vectors are adeno-associated vectors, such as AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV- 10, AAV-11, AAV-12, or any engineered or recombinant AAV known in the art. In a particularly preferred embodiment, the vector is a recombinant AAV-2 (rAAV2).

  In some embodiments, a recombinant adeno-associated virus (rAAV) vector is used to improve target cells, such as retinal bipolar cells (eg, on or off retinal bipolar cells; rod and cone bipolar cells). Includes capsid proteins with mutated tyrosine residues that allow them to have a transduction efficiency. In some cases, rAAV further comprises a promoter (eg, mGluR6 or a fragment thereof) capable of driving expression of a protein of interest in a target cell.

  In one embodiment, mutations may occur at any one or more tyrosine residues of capsid proteins of AAV 1-12 or hybrid AAV. In a specific embodiment, these are surface exposed tyrosine residues. In related embodiments, the tyrosine residue is part of a VP1, VP2 or VP3 capsid protein. In an exemplary embodiment, the mutation may occur at one or more of the following amino acid residues of the AAV-VP3 capsid protein: Tyr252, Tyr272, Tyr444, Tyr500, Tyr700, Tyr704, Tyr730; Tyr275, Tyr281, Tyr508, Tyr576, Tyr612, Tyr673, or Tyr720. Exemplary mutations are tyrosine to phenylalanine mutations, including but not limited to Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F. . In a specific embodiment, these mutations occur in AAV2 serotypes. In some cases, the AAV2 serotype contains the Y444F mutation and / or the AAV8 serotype contains the Y733F mutation, and 444 and 733 indicate the location of the tyrosine point mutation of the viral capsid. In a further embodiment, such mutated AAV2 and AAV8 serotypes encode a light sensitive protein and also comprise a mGluR6 promoter modified to drive expression of such light sensitive protein. Such AAV vectors are described, for example, in Petrs-Silva et al., MoI Ther., 2011 19: 293-301.

  In some embodiments, the expression of the therapeutic transgene is driven by a constitutive promoter, ie, CAG promoter, CMV promoter, LTR. In another embodiment, the promoter is an inducible or cell specific promoter. Cell type specific promoters that allow transgene expression in specific subpopulations of cells, ie, retinal neuronal cells or modified cells, may be preferred. These cells include retinal ganglion cells, photoreceptor cells, bipolar cells, rodoid bipolar cells, on-type pyramidal bipolar cells, retinal ganglion cells, photosensitive retinal ganglion cells, horizontal cells, axillary cells, It may include, but is not limited to, AII axillary cells, or retinal pigment epithelial cells. Cell type specific promoters are well known in the art. Particularly preferred cell type specific promoters include, but are not limited to, mGluR6, NK-3, and Pcp2 (L7). Cell type specific promoters modified using recombinant DNA techniques known in the art to increase expression efficiency and selective targeting are also encompassed by the present invention. For example, the modified mGluR6 promoter contains a combination of regulatory elements from the mGluR6 gene, as described in US Provisional Patent Application No. 61 / 951,360, which is incorporated herein by reference in its entirety.

  In one aspect of the invention, the therapeutic transgene can be any photosensitive opsin. The opsin family of genes includes vertebrate (animal) and invertebrate opsin. Animal opsin is a G-protein coupled receptor (GPCR) with a seven-transmembrane helix that modulates the activity of ion channels. The invertebrate rhodopsin is not usually a GPCR but is a light sensitive or light activated ion pump or ion channel.

  As referred to herein, the opsin gene or light sensitive protein is a channelrhodopsin, ie, a channel opsin (ie, ChR1, ChR2, vChR1, vChR2 from Volvox carteri, and any vertebrate, invertebrate, or Other variants identified from microorganisms), including, but not limited to, halorhodopsin (NpHR), melanopsin, pineal opsin, photopsin, bacteriorhodopsin, proteorhodopsin, and functional variants or chimeras thereof. The photosensitive proteins of the present invention can be naturally present in plant cells, animal cells, archaebacterial cells, algal cells, or bacterial cells, or can be produced through experimental techniques. Examples of opsin genes are discussed in more detail below.

  Examples of channelrhodopsin, that is, channel opsin as transgenes in the present invention are channelrhodopsin Chop1 (also known as ChR1) (GenBank accession numbers AB058890 / AF385748) and Chop2 (also known as ChR2) (GenBank Accession Nos. AB 058 891 / AF 461397), which are two rhodopsins from the green alga Chlamydomonas reinhardtii (Nagel, 2002; Nagel, 2003). Channel opsin is a seven-transmembrane domain protein that is switchable (photosensitive) by light when bound to the chromophore all-trans retinal. When channel opsin is linked to a retinal molecule via a Schiff base bond, it forms a light-gated, non-specific inward rectifying cation channel called channelrhodopsin. These light sensitive channels, when expressed and activated in neural tissue, allow cells to depolarize when stimulated with light (Boyden, 2005). The Chop2 fragment (315 amino acids) has been shown to efficiently increase photosensitivity and visual acuity in mouse models of photoreceptor degeneration (Bi et al., Neuron, 2006 and US Pat. No. 8,470,790; Both are incorporated herein by reference). Chop2 variants and variants as described in PCT Publication WO 2013/134295 (incorporated herein by reference) are also expressed using the promoter described herein. May be The invention also provides the use of the channelrhodopsin of Volvox carteri (ie, vChR1 and vChR2).

  NpHR (Halorhodopsin) (GenBank Accession No. EF 474018) is derived from the haloalkaliphilic archaeon Natronomonas pharaonis. In certain embodiments, variants of NpHR can be generated. In a specific embodiment, single or multiple point mutations to NpHR protein can result in NpHR variants. In a specific embodiment, a mammalian codon optimized version of NpHR can be utilized. In one embodiment, NpHR variants are utilized. In one embodiment, eNpHR (enhanced NpHR) is utilized. Addition of the amino acid FCYENEV to the NpHR C-terminus, together with the addition of a signal peptide from the β subunit of the nicotinic acetylcholine receptor to the NpHR N-terminus, results in the construction of eNpHR.

  Melanopsin (GenBank Accession No. 6693702) is a photopigment found in specialized photosensitive ganglion cells of the retina that is involved in the regulation of circadian rhythm, pupillary light reflex, and other non-visual responses to light is there. In structure, melanopsin is opsin, which is a G protein coupled receptor of the retinylidene protein family. Melanopsin is similar to invertebrate opsin in many respects, including its amino acid sequence and downstream signaling cascades. Melanopsin, like invertebrate opsin, appears to be a bistable photopigment with endogenous photoisomerase activity. In certain embodiments, variants of melanopsin can be generated. In a specific embodiment, single or multiple point mutations to the melanopsin protein can result in melanopsin variants.

  The photosensitive protein may also be at least about 10%, at least about 20%, at least about 25%, any of the photosensitive proteins described herein (ie, ChR1, ChR2, vChR1, vChR2, NpHR, and melanopsin). At least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, It may comprise proteins that are at least about 95%, or at least about 99% identical. The photosensitive protein of the present invention may also include a protein having at least one mutation. The mutation may be a point mutation.

  In some embodiments, light sensitive proteins can modulate signal transduction in neural circuits, and can bidirectionally control the behavior of ionic conductivity at the level of single neurons. In some embodiments, the neurons are retinal neurons, retinal bipolar cells (eg, on or off retinal bipolar cells; rods and pyramidal bipolar cells), retinal ganglion cells, photoreceptor cells, or retinal ataxic Cord cells.

  In some embodiments, poly A chain can be inserted downstream of the transgene in the expression cassette or nucleic acid expression vector of the present invention. Suitable poly A chains are known in the art and include, for example, human growth hormone poly A chain (hGHpA), bovine growth hormone poly A chain (bGHpA), bovine poly A, SV40 poly A, and AV40 pA.

On illumination with a preferred dose of light radiation, the rhodopsin protein opens the channel pore through which H ⁺ , Na ⁺ , K ⁺ , and / or Ca ²⁺ ions flow from the extracellular space into the cell. Activation of rhodopsin channels typically causes depolarization of the cells expressing the channels. The depolarized cells carry information from the rhodopsin expressing cells to other cells of the retina or brain to generate graded and / or action potentials to increase light sensitivity or restore vision. Methods for improving visual acuity or light sensitivity by administration of a vector encoding a channel opsin (or a variant thereof) are described in PCT / US2007 / 068263, the contents of which are incorporated herein in its entirety.

  Thus, a dual rhodopsin system can be used to repeat the on and off pathways essential for visual processing and intelligibility. Briefly, to create the on and off pathways, the Chop2 proteins of the invention can specifically target on-type retinal neurons (ie, on-type ganglion cells and / or on-type bipolar cells) While the hyperpolarised light sensor (ie halorhodopsin or other chloride pump known in the art) targets off-type retinal neurons (ie off-type ganglion cells and / or off-type bipolar cells) be able to. Specific targeting to preferred cell subpopulations can be achieved through the use of different cell type specific promoters. For example, Chop2 expression may be driven by the mGluR6 promoter for target expression in on type retinal neurons (ie, on type ganglion cells and / or on type bipolar cells) while hyperpolarizing channels such as halorhodopsin Is driven by the NK-3 promoter for targeted expression in off-type retinal neurons (ie, off-type ganglion cells and / or off-type bipolar cells).

  An alternative approach to restore the on and off pathways in the retina is achieved by expressing a depolarizing light sensor such as ChR2 in rod bipolar cells or AII axodon. In this approach, depolarization of rodoid dipole cells or AII axodontic cells can result in levels of coneloid bipolar cells and on and off responses downstream of retinal ganglion cells. Thus, the on and off pathways specific to the retina are maintained.

An effective amount of rAAV virion carrying a nucleic acid sequence encoding rhodopsin DNA under control of a selected promoter, preferably a constitutive CMV promoter or a cell specific promoter such as mGluR6, is preferably about 25 to about per injection. It ranges from about 10 ¹⁰ to about 10 ¹³ rAAV infectious units in a volume of between 800 μl. rAAV infectious units can be measured according to McLaughlin, SK et al., 1988, J Virol 62: 1963. More preferably, the effective amount is between about 10 ¹⁰ and about 10 ¹² rAAV infectious units, and the injection volume is preferably between about 50 and about 150 μl. The subject to be treated, preferably within these ranges but possibly outside of it, including other dosages and volumes, including age, weight, general health, and the nature and severity of the particular ocular disorder The physical condition of (preferably human) may be taken into consideration and the expert to be treated may select.

  It may also be desirable to administer additional doses ("boosters") of the subject nucleic acid or rAAV compositions. For example, depending on the duration of transgene expression in the target ocular cell, a second treatment may be administered after 6 months or annually and may be repeated as well. Neutralizing antibodies to AAV are not expected to be generated taking into account the route and dose used, thereby enabling repeated treatment rounds.

  The need for such additional doses can be monitored by a treating professional using, for example, the well-known electrophysiological and other functional tests of retina and vision, and visual behavioral tests. The treating specialist will be able to select the appropriate test, applying conventional techniques in the art. It may be desirable to inject higher volumes of the composition at either single or multiple doses to further improve the relevant outcome parameters.

Eye Disorders Eye disorders for which the methods of the present invention are intended and may be used to improve one or more visual acuity parameters are developmental abnormalities that affect both the anterior and posterior segments of the eye. Including, but not limited to. Anterior disorders include glaucoma, cataract, corneal dystrophy, keratoconus. Disorders of the postpartum include blindness disorder caused by photoreceptor dysfunction and / or death caused by retinal dystrophy and degeneration. Retinal disorders include congenital arrested night blindness, age-related macular degeneration, congenital pyramidal dystrophy, and a large group of retinitis pigmentosa (RP) related disorders. These disorders include genetically predisposed death of photoreceptor cells, rods and cones in the retina, which occur at different ages. Some of these are severe retinopathy such as the subtypes of RP itself that progress with age and cause blindness in childhood and early adulthood, as well as loss of vision early in childhood, as in the first year of life There are RP related diseases such as genetic subtypes of LCA that frequently lead to. The latter disorder is generally characterized by a significant reduction, often complete loss of photoreceptors, rods and cones. Other ocular diseases that may benefit from the methods described herein include retinoblastoma, intraocular melanoma, diabetic retinopathy, hypertensive retinopathy, any inflammation of ocular tissue (ie, choroid retina) Of inflammation, scleritis, keratitis, uveitis etc.) or infections (ie, bacterial or viral) but not limited thereto.

  In particular, virus-mediated delivery of rhodopsin using the method of the present invention results in long-term preservation of ocular tissue structure despite loss of function in the subject's eye cells, and loss of function and normal gene Useful for treatment and / or at least partial visual acuity recovery for subjects who have lost vision due to an eye disorder such as RPE related retinopathy characterized by an association with a defect or absence . A variety of such eye disorders are known, such as childhood onset blindness disease, retinitis pigmentosa, macular degeneration, and diabetic retinopathy, and eye blindness diseases known in the art. It is anticipated that these other disorders and blindness disorders of currently unknown causality, characterized later by the same explanations as above, may also be successfully treated by the methods described herein. Thus, particular ocular disorders treated by the present invention may include the disorders described above, and a number of diseases that have not yet been characterized as such.

Recovery of Photosensitivity These methods described herein may be used in subjects with normal vision and / or impaired vision. Enhanced delivery of therapeutic compounds as described herein may preserve, improve or restore vision. The term "vision" as used herein is defined as the ability of an organism to usefully detect light as a stimulus for identification or action. Vision is intended to encompass the following:
1. Light detection or cognition-the ability to discern whether light is present or not;
2. Light projection-the ability to discern the direction in which the light stimulus is coming;
3. Resolution-the ability to detect different brightness levels (ie contrast) at the grid or text target;
Four. Recognition-The ability to recognize the shape of the visual target by referencing different contrast levels within the target.
Thus, "eyesight" includes the ability to simply detect the presence of light. The methods of the invention can be used to improve or restore visual acuity, such improvement or recovery in visual acuity being, for example, increased light detection or cognition, increased light sensitivity or light sensitivity in response to light stimulation. Increase the ability to discern the direction in which the light stimulus is coming, increase the ability to detect different levels of brightness, increase the ability to recognize the shape of the visual target, and increase the visual evoked potential or transmission from the retina to the cortex. Including. As such, the improvement or restoration of vision may or may not include complete restoration of vision, ie, the vision of the patient treated with the present invention is of the eyesight of an unaffected individual. Recovered to a degree. The restoration of vision described in animal studies below is for the human vision function by increasing one aspect of vision (ie light sensitivity or visual evoked potential) without restoring complete vision. There is a possibility to position a human at the lower end of the. Nevertheless, these individuals can be trained in mobility and potentially in lower order resolution tasks, which are believed to provide a significantly improved level of visual independence compared to full blindness Therefore, positioning at such a level would be a significant benefit. Even basic light perception can be used by visually impaired individuals to achieve specific daily tasks and improve overall mobility, possibilities and quality of life. The compositions and methods are used to improve their vision.

The degree of visual acuity recovery can be determined, for example, through administration of a vector containing DNA encoding a therapeutic transgene such as Chop2 or halorhodopsin or both, and preferably through measurement of visual acuity. Visual acuity can be measured using any of a number of methods well known in the art or methods that have not yet been established. The vision improved or restored by the present invention can be measured by any of the following visual responses:
1. Light detection response by the subject after exposure to a light stimulus, for which evidence is sought for reliable response of the indication or movement in the general direction of the light by the subject individual when illuminated.
2. Light projection response by the subject after exposure to a light stimulus, for which evidence is sought for reliable response of an indication or movement in a specific direction of light by the individual when illuminated.
3. Light resolution by a subject with a visual pattern of light pattern versus dark pattern, which measures the subject's ability to resolve a visual pattern with light pattern versus dark pattern, as evidenced by:
a. Demonstrate target tracking (see above), presence of demonstrable and reliable gaze-like motions such as nystagmus-like eye movements and / or related head or body movements, and / or
b. The presence of a credible ability to distinguish pattern visual stimuli and to indicate such distinctions by verbal or non-verbal means, including, for example, pointing or pushing a bar or button; or
Four. Electrical recording of the visual cortical response to a light flash stimulus or a patterned visual stimulus, also called the visual evoked potential (VEP), the end point of electrical transmission from the restored retina to the visual cortex. The measurement may be by electrical recordings on the surface of the scalp, areas of the surface of the cortex, and / or recordings in cells of the visual cortex.

  Thus, the improvement or restoration of vision according to the present invention may be achieved by increasing the amplitude or dynamics of the photocurrent or electrical response in response to light stimulation in retinal cells, increasing the photosensitivity of retinal cells (ie light in response to light stimulation). Reduce the threshold light intensity required to initiate an electrical current or electrical response, thereby requiring less or less light to induce photocurrent), light induced spiking or spikes It can include, but is not limited to, an increase in light response to the visual cortex, including an increase in the number or amplitude of firings, an increase in the visual evoked potential transmitted from the retina or retinal cells to the visual cortex or the brain.

  Both in vitro and in vivo studies, including recognized animal models of human eye disorders leading to blindness, may be used to assess various parameters of the invention. A number of animal models of human retinopathy, such as childhood blindness, are useful. The examples provided herein allow one of ordinary skill in the art to readily predict that the present methods can be similarly used to treat a range of retinal diseases.

  Although previous studies by others have demonstrated that retinal degeneration can be delayed by gene therapy techniques, the present invention is expected to produce or improve various parameters of visual acuity, including behavioral parameters Demonstrate, clear physiological repair of function.

  Behavioral measures can be obtained using known animal models and tests, for example, movements in the water maze, where subjects whose vision is stored or restored to varying degrees will swim towards the light (Hayes, JM et al., 1993, Behav Genet 23: 395-403).

  In models where blindness is induced during adulthood or congenital blindness occurs slowly enough to experience before the individual loses vision, the subject may be trained in various trials. Thus, when re-applying these tests after loss of vision to test the efficacy of the present compositions and methods for their visual acuity-restoring effect, the animals do not learn the task anew during the blinding state May be Other behavioral tests do not require learning and rely on the instinct of certain behaviors. An example is the gaze movement nystagmus test (Balkema GW et al., 1984, Invest Ophthalmol Vis Sci. 25: 795-800; Mitchiner JC et al., 1976, Vision Res. 16: 1169-71).

  The invention may also be used in combination with other forms of vision treatment known in the art to improve or restore vision. For example, the use of visual prostheses, including retinal implants, cortical implants, lateral knee implants, or optic nerve implants. Thus, in addition to the genetic modifications that make retinal neurons survive using the present methods, the subject to be treated may be provided with a visual prosthesis before, simultaneously or after the molecular method is used. The effectiveness of the visual prosthesis can be improved with training of the individual, thus enhancing the potential impact of Chop2 transformation of patient cells as contemplated herein. The training method is intended to recognize (i) various levels of light and / or pattern stimulation, and / or (ii) environmental stimuli from common light sources or objects that will be understood by those skilled in the art. Training, and visually detecting local objects and training the objects to move between the objects more efficiently than without training. Training of orientation and movement. In fact, any visual stimulation techniques typically used in the field of low vision rehabilitation are applicable here.

  As used herein, an "subject" means an individual. Thus, "subject" refers to domesticated animals (eg, cats, dogs etc.), livestock (eg, cows, horses, pigs, sheep, goats etc.), experimental animals (eg, mice, rabbits, rats, guinea pigs etc.) And birds can be included. "Subject" can also include mammals such as primates or humans. Preferably, the subject is a human. A "subject in need" is a subject suffering from, or at risk of developing or suffering from an eye disease or disorder. Subjects at risk of developing or suffering from an eye disease or disorder can be diagnosed by a physician or eye specialist using routine methods in the art.

Example 1 Plasmin Increases Delivery of AAV2 Vector Encoding Chop2 to the Retina Delivery of a therapeutic virus construct encoding a functional GFP-channel opsin-2 protein to the retina was examined. Injection of 6 × 10 ¹² vg / ml of AAV2 vector AAV2 / 2-ChR2-GFP-WPRE-hGHpA in control (FIG. 1A) or co-injection with plasmin (FIG. 1B), one month old C56BL / 6J eyes Into the vitreous cavity of The concentration of plasmin injected with the vector was 0.025 IU / eye. One month later, mouse retinas were isolated and retinal vertical sections were prepared. Sections were immunostained to count cells. Co-injection with plasmin was shown to increase the transduction efficiency of the therapeutic AAV2-ChR2-GFP vector, as evidenced by immunofluorescence analysis of the sections, as evidenced by the increased fluorescence compared to the control ( Figure 1).

Example 2 Plasmin Increases Transduction Efficiency in Retinal Ganglion Cells Using the same experimental setup as in Example 1, a vector encoding GFP (2 × 10 ¹² vg / ml) was used as a control or a variety of Co-injected with any of the concentrations plasmin: low (L = 0.005 IU / eye), medium (M = 0.025 IU / eye), and high (H = 0.100 IU / eye). One month later, whole retinas were prepared and immunostained. Representative images for each plasmin concentration and control are shown in FIG. As shown, treatment with plasmin increases GFP expression.

  To further quantify these results, GFP expressing retinal ganglion cells were counted from multiple unit areas of 223 μm × 167 μm. The results are shown in FIG. 3A. As shown, treatment with low, medium and high doses of plasmin resulted in statistically significantly increased levels of GFP expression in retinal ganglion cells.

  Neurotoxicity as a result of plasmin injection was also examined. Whole retinas were stained with DAPI for cell counting. The number of cells over multiple unit areas of 223 μm × 167 μm was counted and compared between control, low dose, medium dose, and high dose plasmin. As shown in FIG. 3B, cell numbers were not found to be significantly different between control and plasmin treated retinas (p = 0.74). As such, the concentrations of plasmin tested were not shown to have any neurotoxic effect on retinal ganglion cells, thereby demonstrating that plasmin is safe for use in the eye, even at high doses.

Example 3 Plasmin Increases Transduction Efficiency in Retinal Bipolar Cells A comparison of the transduction efficiency of viral vectors encoding mCherry fluorescent protein when co-injected with different concentrations of plasmin was evaluated in vivo. Specifically, we examined global levels and localization of mCherry expression throughout the retina. An AAV2 vector with Y444F capsid mutation carrying mCherry under the control of the mGluR6 promoter was injected at a concentration of 2 × 10 ¹² vg / ml. The mGluR6 promoter specifically directs mCherry expression to retinal bipolar cells.

  The AAV2 mCherry vector was co-injected with three doses of plasmin: high (H = 0.100 IU / eye), medium (M = 0.025 IU / eye), and low (L = 0.005 IU / eye). One month later, the retina was isolated and the whole retina was prepared. Transduction efficiency was assessed by immunostaining of mCherry for immunofluorescence analysis and cell counting. Cells were counted from multiple unit areas of 223 μm × 167 μm.

  Injection of the vector without plasmin did not result in uniform mCherry expression in retinal bipolar cells across the entire retina (FIG. 4, control, upper panel). Transduction efficiency was low in the central (A) and middle (B) retinas but high in the periphery. Co-injection (low, medium and high, lower panels) of increasing doses of AAV2 mCherry vector with plasmin resulted in an increase in transduction efficiency in each retinal area in a dose dependent manner.

  Quantitative results from immunofluorescence images were verified by cell counting. Quantification of mCherry-expressing cells showed that plasmin significantly increases the density of mCherry-expressing retinal cells when co-injected with or without plasmin. The increase in transduction efficiency with plasmin compared to control was statistically significant at all three doses of plasmin at the center of the retina. Intermediate and high doses of plasmin resulted in a statistically significant increase in mCherry expression in and around the middle region of the retina. These results indicate that plasmin enhances transduction efficiency throughout the retina, including the periphery, middle and central regions of the retina.

Other Embodiments While the present invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to exemplify and limit the scope of the present invention as defined by the appended claims. Not intended. Other aspects, advantages, and modifications are within the scope of the following claims.

  The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference.

  While the present invention has been particularly shown and described with respect to its preferred embodiments, various modifications in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims. It will be understood by one skilled in the art that it may be done.

Claims (20)

  A pharmaceutical composition for enhancing delivery of a therapeutic agent to a subject's eye, comprising plasmin or a derivative thereof, used in combination with the therapeutic agent, wherein the therapeutic agent is a nucleic acid expression vector Composition.   The pharmaceutical composition according to claim 1, wherein plasmin or a derivative thereof is miniplasmin or microplasmin (occliplasmin).   The pharmaceutical composition according to claim 1, wherein the nucleic acid expression vector is a viral vector containing a transgene.   5. The pharmaceutical composition of claim 3, wherein the transgene is opsin.   5. The pharmaceutical composition of claim 4, wherein the opsin is selected from the group consisting of channelrhodopsin, halorhodopsin, melanopsin, pineal opsin, bacteriorhodopsin, and proteorhodopsin, or a functional variant thereof.   6. The pharmaceutical composition of any one of claims 3-5, wherein the transgene is operably linked to a cell specific promoter.   The pharmaceutical composition according to any one of claims 1 to 6, wherein the therapeutic agent is encapsulated in nanoparticles, polymers or liposomes.   The pharmaceutical composition according to claim 1, wherein the subject suffers from an eye disease or disorder.   The pharmaceutical composition of claim 1 suitable for simultaneous or sequential delivery with a therapeutic agent.   The pharmaceutical composition of claim 1, wherein the therapeutic agent is suitable for delivery to retinal cells. 11. The pharmaceutical composition according to claim 10 , wherein the retinal cells are retinal ganglion cells, retinal bipolar cells, retinal horizontal cells, axillary cells, photoreceptor cells, Mueller glial cells, or retinal pigment epithelial cells.   The pharmaceutical composition of claim 1 suitable for delivery to the vitreous of the eye.   A pharmaceutical composition for increasing light sensitivity or improving or restoring visual acuity in a subject, comprising plasmin or a derivative thereof, a nucleic acid encoding opsin which is suitable for delivery to the vitreous of the eye Pharmaceutical composition for use in combination with an expression vector. 14. The pharmaceutical composition of claim 13 , wherein the opsin is selected from the group consisting of channelrhodopsin, halorhodopsin, melanopsin, pineal opsin, bacteriorhodopsin, and proteorhodopsin, or a functional variant thereof. The pharmaceutical composition according to claim 13 or 14 , wherein the subject has an eye disease or disorder. 14. The pharmaceutical composition of claim 13 , wherein the opsin is operably linked to a cell specific promoter. The pharmaceutical composition according to claim 13 , which is suitable for simultaneous or sequential delivery with a nucleic acid expression vector. 14. The pharmaceutical composition of claim 13 , wherein the nucleic acid expression vector is suitable for delivery to retinal cells. 19. The pharmaceutical composition according to claim 18 , wherein the retinal cells are retinal ganglion cells, retinal bipolar cells, retinal horizontal cells, axillary cells, photoreceptor cells, Mueller glial cells, or retinal pigment epithelial cells. 14. The pharmaceutical composition of claim 13 , wherein the nucleic acid expression vector is a viral vector.

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